DMD_D.2.2.002
Please quote this SOP in your methods.
Measuring Isometric Force/Torque Decrement after Eccentric
Contraction in Vivo
SOP (ID) Number
DMD_D.2.2.002
Version
2.0
Issued
November 14th, 2008
Last reviewed
June 30th, 2010; January 31, 2013; June 13, 2015
Author
Joe Kornegay
College of Veterinary Medicine and Biomedical
Sciences, Texas A&M University, College Station, TX
USA
Working group
members
Robert Grange (Virginia Tech, Department of Human
Nutrition, Foods and Exercise, Blacksburg, VA, USA )
George Carlson (Department of Physiology, A.T. Still
University, Kirksville, USA)
Stephane Blot (Veterinary School of Medicine, Maison
Alfort, France)
Margot Coville (INSERM, Nantes)
SOP responsible
Joe Kornegay
Official reviewer
Robert Grange
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DMD_D.2.2.002
TABLE OF CONTENTS
OBJECTIVE.................................................................................................................................. 3
SCOPE AND APPLICABILITY ........................................................................................................ 3
CAUTIONS .................................................................................................................................. 3
MATERIALS ................................................................................................................................ 3
METHODS .................................................................................................................................. 4
EVALUATION AND INTERPRETATION OF RESULTS .................................................................... 6
REFERENCES .............................................................................................................................. 8
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DMD_D.2.2.002
1.
OBJECTIVE
Dystrophin serves to buttress the muscle cell membrane. In the absence of dystrophin,
the membrane is prone to tearing during minimal exercise. Dystrophic muscles are
particularly prone to injury subsequent to eccentric (lengthening) contractions.1 As an
example, mdx mice have near-normal absolute force measurements but demonstrate
greater-than-normal force decrement after eccentric muscle contractions. 2 We have
previously shown that GRMD dogs also have a greater-than-normal force decrement with
eccentric contractions.3,4
2.
SCOPE AND APPLICABILITY
As discussed in the GRMD tibiotarsal joint torque protocol, phenotypic variation in
affected dogs confounds statistical analysis, necessitating larger group sizes to show
significance. With localized treatments, the effect of phenotypic variation is less of a
concern because the untreated opposite limb can serve as the control. With systemic
treatments, baseline measurements should be established prior to treatment so that each
dog serves as its own control. Evaluation of the force decrement incurred due to eccentric
muscle contraction offers another option by which each dog can be used as its own control.
3.
CAUTIONS
Dogs must be anesthetized. A nerve stimulator, specialized, custom-made stereotactic
frame, and servomotor are required.
See METHODS and EVALUATION AND
INTERPRETATION OF RESULTS (below) for guidance on interpretation.
4.
MATERIALS
Separate personnel are utilized for anesthetic management and force measurements.
Dogs are anesthetized and positioned in dorsal recumbency with one of the pelvic limbs
placed in a custom-made stereotactic frame such that the hip (coxofemoral), knee (stifle),
and hock (tibiotarsal) joints are all at 90 o angles (see GRMD tibiotarsal joint force protocol
and Figure 1). In an initial study, eccentric contractions were induced in flexor muscles of
the cranial tibial compartment by stimulating the sciatic trunk in the mid-thigh area.3 This
caused contraction of both the tibiotarsal joint flexors and extensors. Because the extensors
are more powerful, eccentric (lengthening) contractions were induced in the flexors.
More recently, we have developed a technique whereby the common peroneal nerve
is stimulated while simultaneously extending the tibiotarsal joint with a servomotor (Aurora
Scientific, Ontario, Canada) coupled to a lever arm. The servomotor extends (lengthens)
muscles of the cranial tibial compartment. Movement of the lever arm is controlled by use
of a computer and customized LabView software (Aurora Scientific). Before beginning the
experimental contractions, isometric forces from single twitches are measured. The
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DMD_D.2.2.002
common peroneal nerve is stimulated with optimally-positioned percutaneous electrodes
(Model S48 Solid State Square Wave Stimulator, Grass Instruments, Quincy, MA, USA).
Voltage is adjusted until twitch force (Pt) reaches a maximum. To maximally activate the
muscles of interest to produce a tetanus, stimulation voltage is 50% greater than necessary
to achieve Pt. Stretches during tetanic stimulation are performed 10 times, once every 5
sec. Following a 4 minute rest, the series of 10 stretches is repeated. Thus, the muscles of
the cranial tibial compartment are repeatedly stretched to induce mechanical damage.
5.
METHODS
5.1. Anesthetic protocol (Note, in a preliminary study, mean alveolar concentration [MAC]
values for isoflurane did not significantly affect force measurement values [Schueler RO,
Koch J, Kornegay JN, unpublished data]).
20-30 minutes prior to anesthesia induction:
 Pre-anesthetic agents:
- Atropine sulfate (0.04 mg/kg, IM)
- Acepromazine maleate (0.02 mg/kg, IM) for dogs weighing greater than 5 kg
- Butorphanol tartrate (0.4 mg/kg, IM)
Anesthetic induction:
 Anesthetic agents:
- Propofol (up to 3 mg/kg, IV – slowly!) for dogs weighing greater than 5 kg
- Isoflurane or sevoflurane (to effect, inhaled) (avoid masking down)
Anesthetic monitoring:
During anesthesia, ECG, heart and respiratory rate, blood pressure, end tidal (Et)CO 2, and
saturation of hemoglobin by peripheral oxygen (SpO 2) are monitored continuously with a
pulse oximeter (Cardell Multiparameter Monitor 9405, Minrad International, Inc, Orchard
Park, NY). These values, as well as capillary refill time and anesthetic setting, are recorded
every 15 minutes.
Anesthetic recovery:
Monitor affected and carrier dogs closely during anesthetic recovery until fully awake and in
sternal recumbency.
- Naloxone (up to 0.4 mg/kg, SQ) for rapid recovery; given in ½ dose increments (1 st
dose given while the dog is still intubated and breathing O 2; 2nd dose, if necessary,
after extubated and/or if respiration drops below 7 breaths per minute).
5.2. Position the dog in dorsal recumbency.
5.3. Measure the tibiotarsal joint angle (see separate GRMD protocol).
5.4. Calibrate the force/torque transducer before each set of measurements.
5.5. Place one of the pelvic limbs in a stereotactic frame such that the hip (coxofemoral),
knee (stifle), and hock (tibiotarsal) joints are all at 90 o angles (see GRMD tibiotarsal joint
torque force protocol).
5.6. Palpate the common peroneal nerve just distal to the head of the fibula as it courses
across the bone. Paired stimulating and reference 27-gauge monopolar needle electrodes
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DMD_D.2.2.002
are placed subcutaneously on either side of the nerve (Figure 1A). The electrodes are
optimally positioned and the voltage is adjusted until twitch force (Pt) reaches a maximum.
To maximally activate the muscles of interest to produce a tetanus, stimulation voltage is
50% greater than necessary to achieve Pt. (generally, supramaximal pulses of 150 V are
applied). An electromyography (EMG) unit (TE42, Teca Corp, Pleasantville, NY, USA) can be
used to record electrical activity in tibiotarsal joint flexor muscles (cranial tibialis and long
digital extensor) to ensure proper needle placement. Eccentric contractions are induced
using square wave pulses of 100 μsec. The contraction is held isometric at the optimal fiber
Figure 1. A. Left pelvic limb of a 6-month-old GRMD dog immobilized in a
stereotactic frame, with needles positioned to stimulate the peroneal nerve. B.
Histogram showing mean torque (N-m) generated by tetanic tibiotarsal joint flexion
during a series of 10 tetanic contractions (50 pps, pulse width 100 μsec, duration
1.7 sec, each being separated by 5 secs). Near the conclusion of each tetanic
contraction, these maximally-stimulated cranial tibial compartment muscles were
forcibly stretched by the servomotor as the tibiotarsal joint was extended a further
30 degrees at a rate of 290 degrees/sec (~ 0.7 muscle length/sec) (see D). The
mean value of the initial contractions (blue bar) was ~ 1.4 N-m, while the tenth
contraction (red bar) was ~ 0.2 N-m, representing an 85% decrement. C. The
characteristic isometric tetanic mechanical potential produced by tibiotarsal joint
flexors is illustrated. D. A single tetanic mechanical potential analogous to that
seen in C is followed by a sharp further deflection reflecting the eccentric
contraction induced by the servomotor, with an immediate return to baseline. The
baseline deflection following the potential is due to vibration artifact. Note, data
shown here were collected using a modification of the method described. The
tetanic run was 700 msec, with the first 500 isometric followed by 200 msec of
stretch. From reference 5.
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DMD_D.2.2.002
The muscles of the cranial tibial compartment are stretched by the servomotor at a rate of
0.7 muscle lengths/sec, such that the muscles are stretched to 107% of L o. Stretches are
performed 10 times, once every 5 sec. Following a 4 minute rest, the series of 10 stretches
is repeated. Thus, the muscles of the cranial tibial compartment are repeatedly stretched to
induce mechanical damage. Torque is recorded by the computer software. Contractioninduced injury is quantified by the force deficit (P d), which is defined as the percent
difference between maximum isometric force (Po) before and after exercise (in this case,
eccentric stretches). Force deficit is expressed as a percent change of the initial value of P o,
using the following equation:
Force deficit (Pd) = [Po - Po after stretch/Po] X 100
6.
EVALUATION AND INTERPRETATION OF RESULTS
Based on results from mdx mice, we expected that GRMD dogs would have a greater
decrement in force following eccentric contractions of muscles of the cranial tibial
compartment. In an initial study, GRMD dogs (n = 10) had a greater decrement (- 63% ± 4; p
< 0.0001) than either carrier (n = 7; - 13 ± 1) or normal (n = 5; - 8 ± 3) dogs after a series of 30
eccentric contractions administered in three groups of 10 (see technique above) (Figure 2).
However, in subsequent studies, we have found that the decrement in normal dogs can be ~
25%, approaching that of GRMD dogs (Kornegay JN, unpublished). It may be useful to
compare data from the first 10 eccentric contractions alone, since the differential is greater
at that point. Data also still must be correlated with histologic markers such as Evans blue
dye exclusion before definitive conclusions can be drawn.
Figure 2. Representative torque tracings (left) during 10 ECCs (darkest line = 1 st and lightest
line = 10th contractions). Contraction torque rapidly decrements (middle) in GRMD dogs
(triangles, bottom trace) rapidly compared to normal (diamonds, top trace) and carrier
(squares, middle trace) dogs. Broken lines through the x-axis indicate a 4-min rest period.
After 30 contractions (right), GRMD dogs have lost 63% of their original torque, whereas
carriers lost 13% and normals lost 8%. Black = normal; gray = carrier; light = GRMD.
*Significantly different (one-way ANCOVA) compared to the other genotypes (P < 0.05).
From reference 4.
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Potential Advantages/Disadvantages of the Methodology
Advantages
As with the tibiotarsal joint torque method, this technique allows serial, non-invasive
evaluation of contractile force and eccentric contraction decrement. The effects of
phenotypic variation are offset because the force decrement is individualized for each dog.
Disadvantages
Instrumentation is more expensive and must be customized. Muscles of the cranial
tibial compartment may undergo functional hypertrophy subsequent to initial injury. Thus,
it is not clear that functional test results will accurately predict overall response to
treatment. As an example, functional hypertrophy may be associated with deposition of
dystrophin surrogates that could, in principle, protect muscles from eccentric contraction.
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7. REFERENCES
1.
Edwards RHT, Jones DA, Newham DJ, Chapman SJ: Role of mechanical damage in the
pathogenesis of proximal myopathy in man. Lancet 1:548-552, 1984.
2.
Moens P, Baatsen PHWW, Marechal G: Increased susceptibility of EDL muscles from
mdx mice to damage induced by contractions with stretch. J MusRes Cell Motil
14:446-451, 1993.
3.
Childers MK, Okamura CS, Bogan DJ, Bogan JR, Petroski GF, McDonald K, Kornegay JN:
Eccentric contraction injury in dystrophic canine muscle. Arch Phys Med Rehabil
83:1572-1578, 2002.
4.
Tegler CJ, Grange RW, Bogan DJ, Markert CD, Case D, Kornegay JN, Childers MK:
Eccentric contractions induce rapid isometric torque drop in dystrophic-deficient
dogs. Muscle Nerve 42:130-132, 2010.
5.
Kornegay JN, Bogan JR, Bogan DJ, Childers MK, Li J, Nghiem P, Detwiler DA, Larsen CA,
Grange RW, Bhavaraju-Sanka RK, Tou S, Keene BP, Howard JF, Jr, Wang J, Fan Z,
Schatzberg SJ, Styner MA, Flanigan KM, Xiao X, Hoffman EP: Canine models of
Duchenne muscular dystrophy and their use in therapeutic strategies. Mamm
Genome 23:85-108, 2012.
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